Methods and compositions for treating hemophilia

ABSTRACT

Disclosed herein are methods and compositions for insertion of transgene sequences encoding proteins involved in clotting into the genome of a cell for treating conditions including hemophilias.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/673,163, filed Aug. 9, 2017, now U.S. Pat. No. 10,407,476,which is a continuation of U.S. patent application Ser. No. 14/564,722,filed Dec. 9, 2014, now U.S. Pat. No. 9,771,403, which claims thebenefit of U.S. Provisional Application No. 61/913,838, filed Dec. 9,2013 and U.S. Provisional Application No. 61/943,884, filed Feb. 24,2014, the disclosures of which are hereby incorporated by reference inits entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 26, 2019 isnamed 83250115US5SL.txt and is 13,401 bytes in size.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present disclosure is in the fields of gene modification andtreatment of hemophilia.

BACKGROUND

Hemophilias such as hemophilia A and hemophilia B, are genetic disordersof the blood-clotting system, characterized by bleeding into joints andsoft tissues, and by excessive bleeding into any site experiencingtrauma or undergoing surgery. Hemophilia A is clinicallyindistinguishable from hemophilia B, but Factor VIII (FVIII or F8) isdeficient or absent in hemophilia A while Factor IX (FIX or F.IX) isdeficient or absent in patients with hemophilia B. The Factor VIII geneencodes a plasma glycoprotein that circulates in association with vonWilebrand's factor in its inactive form. Upon surface injury, theintrinsic clotting cascade initiates and factor VIII is released fromthe complex and becomes activated. The activated form acts with FactorIX to activate Factor X to become the activated Xa, eventually leadingto change of fibrinogen to fibrin and clot induction. See, Levinson etal. (1990) Genomics 7(1):1-11. 40-50% of hemophilia A patients have achromosomal inversion involving Factor VIII intron 22 (also known asIVS22). The inversion is caused by an intra-chromosomal recombinationevent between a 9.6 kb sequence within the intron 22 of the Factor VIIIgene and one of the two closely related inversely orientated sequenceslocated about 300 kb distal to the Factor VIII gene, resulting in aninversion of exons 1 to 22 with respect to exons 23 to 26. See, Textbookof Hemophilia, Lee et al. (eds) 2005, Blackwell Publishing. Otherhemophilia A patients have defects in Factor VIII including active sitemutations, and nonsense and missense mutations. For its part, Factor IX(F.IX or FIX) encodes one of the serine proteases involved with thecoagulation system, and it has been shown that restoration of even 3% ofnormal circulating levels of wild type Factor IX protein can preventspontaneous bleeding. Additional hemophilias are associated withaberrant expression of other clotting factors. For example, Factor VIIdeficiency is an autosomal recessive trait occurring in approximately 1in 300,000 to 500,000 people and is associated with inadequate FactorVII levels in the patient. Similarly, Factor X deficiency is also anautosomal recessive trait occurring in 1 in every 500,000 to 1 millionpeople, and is caused by genetic variants of the FX gene. Factor Xdeficiency can have varying degrees of severity in the patientpopulation.

Current treatments for Hemophilia B rely on chronic, repeatedintravenous infusions of purified recombinant Factor IX and suffer froma number of drawbacks. This includes the need for repeated intravenousinfusions, is associated with inhibitor formation, and is prophylacticrather than curative.

Gene therapy for patients with Hemophilia A or B, involving theintroduction of plasmid and other vectors (e.g., AAV) encoding afunctional Factor VIII or Factor IX proteins have been described. See,e.g., U.S. Pat. Nos. 6,936,243; 7,238,346; and 6,200,560; Shi et al.(2007) J Thromb Haemost. (2):352-61; Lee et al. (2004) Pharm. Res.7:1229-1232; Graham et al. (2008) Genet Vaccines Ther. 3:6-9; Manno etal. (2003) Blood 101(8): 2963-72; Manno et al. (2006) Nature Medicine12(3): 342-7; Nathwani et al. (2011) Molecular Therapy 19(5): 876-85;Nathwani et al. (2011); N Engl J Med. 365(25): 2357-65. However, inthese protocols, the formation of inhibitory anti-factor VIII or IX(anti-F8 or anti-FIX) antibodies and antibodies against the deliveryvehicle remains a major complication of Factor VIII and Factor IXreplacement-based treatment for hemophilia. See, e.g., Scott & Lozier(2012) Br J Haematol. 156(3):295-302.

Various methods and compositions for targeted cleavage of genomic DNAhave been described. Such targeted cleavage events can be used, forexample, to induce targeted mutagenesis, induce targeted deletions ofcellular DNA sequences, and facilitate targeted recombination at apredetermined chromosomal locus. See, e.g., U.S. Pat. Nos. 8,623,618;8,034,598; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558;7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925;8,110,379; 8,409,861; U.S. Patent Publication Nos. 2003/0232410;2005/0208489; 2005/0026157; 2006/0063231; 2008/0159996; 2010/00218264;2012/0017290; 2011/0265198; 2013/0137104; 2013/0122591; 2013/0177983;2013/0177960 and 2015/0056705, the disclosures of which are incorporatedby reference in their entireties for all purposes. These methods ofteninvolve the use of engineered cleavage systems to induce a double strandbreak (DSB) or a nick in a target DNA sequence such that repair of thebreak by an error born process such as non-homologous end joining (NHEJ)or repair using a repair template (homology directed repair or HDR) canresult in the knock out of a gene or the insertion of a sequence ofinterest (targeted integration). This technique can also be used tointroduce site specific changes in the genome sequence through use of adonor oligonucleotide, including the introduction of specific deletionsof genomic regions, or of specific point mutations or localizedalterations (also known as gene correction). Cleavage can occur throughthe use of specific nucleases such as engineered zinc finger nucleases(ZFN), transcription-activator like effector nucleases (TALENs), orusing the CRISPR/Cas system with an engineered crRNA/tracr RNA (singleguide RNA′) to guide specific cleavage. Further, targeted nucleases arebeing developed based on the Argonaute system (e.g., from T.thermophilus, known as ‘TtAgo’, see Swarts et al. (2014) Nature507(7491): 258-261), which also may have the potential for uses ingenome editing and gene therapy.

This nuclease-mediated targeted transgene insertion approach offers theprospect of improved transgene expression, increased safety andexpressional durability, as compared to classic integration approaches,since it allows exact transgene positioning for a minimal risk of genesilencing or activation of nearby oncogenes.

Targeted integration of a transgene may be into its cognate locus, forexample, insertion of a wild type transgene into the endogenous locus tocorrect a mutant gene. Alternatively, the transgene may be inserted intoa non-cognate locus, for example a “safe harbor” locus. Several safeharbor loci have been described, including CCR5, HPRT, AAVS1, Rosa andalbumin. See, e.g., U.S. Pat. Nos. 7,951,925 and 8,110,379; U.S. PatentPublication Nos. 2008/0159996; 2010/00218264; 2012/0017290;2011/0265198; 2013/0137104; 2013/0122591; 2013/0177983, 2013/0177960 and2015/0056705. For example, U.S. Patent Publication No. 2011/0027235relates to targeted integration of functional proteins into isolatedstem cells and U.S. Patent Publication No. 2012/0128635 describesmethods of treating hemophilia B. See also Li et al. (2011) Nature 475(7355):217-221 and Anguela et al. (2013) Blood 122:3283-3287.

However, there remains a need for additional compositions and methods oftreating hemophilias.

SUMMARY

Disclosed herein are methods and compositions for targeted integrationof a sequence encoding a protein, such as a functional clotting factorprotein (e.g., Factor VII, Factor VIII, Factor IX, Factor X, and/orFactor XI). Expression of a functional Factor VIII (“F8”) and/or FactorIX (“F.IX” or “FIX”) protein can result, for example, in the treatmentand/or prevention of hemophilia A (Factor VIII) and/or hemophilia B(Factor IX), while expression of a functional Factor VII or Factor X cantreat or prevent hemophilias associated with Factor VII and/or Factor Xdeficiency. Nucleases, for example engineered meganucleases, zinc fingernucleases (ZFNs), TALE-nucleases (TALENs including fusions of TALEeffectors domains with nuclease domains from restriction endonucleasesand/or from meganucleases (such as mega TALEs and compact TALENs)),Ttago system and/or CRISPR/Cas nuclease systems are used to cleave DNAat a ‘safe harbor’ gene locus (e.g. CCR5, AAVS1, HPRT, Rosa or albumin)in the cell into which the gene is inserted. Targeted insertion of adonor transgene may be via homology directed repair (HDR) ornon-homology repair mechanisms (e.g., NHEJ donor capture). The nucleasecan induce a double-stranded (DSB) or single-stranded break (nick) inthe target DNA. In some embodiments, two nickases are used to create aDSB by introducing two nicks. In some cases, the nickase is a ZFN, whilein others, the nickase is a TALEN or a CRISPR/Cas nickase. In someembodiments, the methods and compositions involve at least one proteinthat binds to an albumin gene in a cell.

In one aspect, described herein is a non-naturally occurring zinc-fingerprotein (ZFP) that binds to a target site in a region of interest (e.g.,an albumin gene) in a genome, wherein the ZFP comprises one or moreengineered zinc-finger binding domains. In one embodiment, the ZFP is azinc-finger nuclease (ZFN) that cleaves a target genomic region ofinterest, wherein the ZFN comprises one or more engineered zinc-fingerbinding domains and a nuclease cleavage domain or cleavage half-domain.Cleavage domains and cleavage half domains can be obtained, for example,from various restriction endonucleases and/or homing endonucleases. Inone embodiment, the cleavage half-domains are derived from a Type IISrestriction endonuclease (e.g., FokI). In certain embodiments, the zincfinger domain recognizes a target site in an albumin gene, for example azinc finger protein with the recognition helix domains ordered as shownin a single row of Table 1. In some embodiments, the zinc finger proteincomprises five or six zinc finger domains designated and ordered F1 toF5 or F1 to F6, as shown in a single row of Table 1.

In another aspect, described herein is a Transcription Activator LikeEffector (TALE) protein that binds to target site in a region ofinterest (e.g., an albumin gene) in a genome, wherein the TALE comprisesone or more engineered TALE binding domains. In one embodiment, the TALEis a nuclease (TALEN) that cleaves a target genomic region of interest,wherein the TALEN comprises one or more engineered TALE DNA bindingdomains and a nuclease cleavage domain or cleavage half-domain. Cleavagedomains and cleavage half domains can be obtained, for example, fromvarious restriction endonucleases and/or homing endonucleases(meganuclease). In one embodiment, the cleavage half-domains are derivedfrom a Type IIS restriction endonuclease (e.g., FokI). In otherembodiments, the cleavage domain is derived from a meganuclease, whichmeganuclease domain may also exhibit DNA-binding functionality.

In another aspect, described herein is a CRISPR/Cas system that binds totarget site in a region of interest (e.g., an albumin gene) in a genome,wherein the CRISPR/Cas system comprises one or more engineered singleguide RNA or a functional equivalent, as well as a Cas9 nuclease.

The nucleases (e.g., ZFN, CRISPR/Cas system, Ttago and/or TALEN) asdescribed herein may bind to and/or cleave the region of interest in acoding or non-coding region within or adjacent to the gene, such as, forexample, a leader sequence, trailer sequence or intron, or within anon-transcribed region, either upstream or downstream of the codingregion. In certain embodiments, the nuclease (e.g., ZFN) binds to and/orcleaves an albumin gene.

In another aspect, described herein is a polynucleotide encoding one ormore nucleases (e.g., ZFNs, CRISPR/Cas systems, Ttago and/or TALENsdescribed herein) or other proteins. The polynucleotide may be, forexample, mRNA. In some aspects, the mRNA may be chemically modified (Seee.g. Kormann et al. (2011) Nature Biotechnology 29(2):154-157). In otheraspects, the mRNA may comprise an ARCA cap (see U.S. Pat. Nos. 7,074,596and 8,153,773). In further embodiments, the mRNA may comprise a mixtureof unmodified and modified nucleotides (see U.S. Patent Publication No.2012/0195936).

In another aspect, described herein is an expression vector (e.g., aZFN, CRISPR/Cas system, Ttago and/or TALEN expression vector) comprisinga polynucleotide, encoding one or more proteins including nucleases(e.g., ZFNs, CRISPR/Cas systems, Ttago and/or TALENs) as describedherein, operably linked to a promoter. In one embodiment, the expressionvector is a viral vector. In one aspect, the viral vector exhibitstissue specific tropism.

In another aspect, described herein is a host cell comprising one ormore expression vectors, including nuclease (e.g., ZFN, CRISPR/Cassystems, Ttago and/or TALEN) expression vectors.

In another aspect, pharmaceutical compositions comprising an expressionvector as described herein are provided. In some embodiments, thepharmaceutical composition may comprise more than one expression vector.In some embodiments, the pharmaceutical composition comprises a firstexpression vector comprising a first polynucleotide, and a secondexpression vector comprising a second polynucleotide. In someembodiments, the first polynucleotide and the second polynucleotide aredifferent. In some embodiments, the first polynucleotide and the secondpolynucleotide are substantially the same. The pharmaceuticalcomposition may further comprise a donor sequence (e.g., a Factor IXdonor sequence). In some embodiments, the donor sequence is associatedwith an expression vector.

In one aspect, the methods and compositions of the invention comprisegenetically modified cells comprising a transgene expressing afunctional version of a protein that is aberrantly expressed in ahemophilia (Factor VII, Factor VIII, Factor IX, Factor X, and/or FactorXI protein), in which the transgene is integrated into an endogenoussafe-harbor gene (e.g., albumin gene) of the cell's genome. In certainembodiments, the transgene is integrated in a site-specific (targeted)manner using at least one nuclease. In certain embodiments, the nuclease(e.g., ZFNs, TALENs, Ttago and/or CRISPR/Cas systems) is specific for asafe harbor gene (e.g. CCR5, HPRT, AAVS1, Rosa or albumin. See, e.g.,U.S. Pat. Nos. 7,951,925 and 8,110,379; U.S. Patent Publication Nos.2008/0159996; 2010/0218264; 2012/0017290; 2011/0265198; 2013/0137104;2013/0122591; 2013/0177983; 2013/0177960; and 2015/0056705. In someembodiments, the safe harbor is an albumin gene.

In another aspect, described herein is a method of genetically modifyinga cell, in vitro and/or in vivo, to produce Factor VII, Factor VIII,Factor IX, Factor X and/or Factor XI, the method comprising cleaving anendogenous safe harbor gene in the cell using one or more nucleases(e.g., ZFNs, TALENs, CRISPR/Cas) such that a transgene encoding FactorVII, Factor VIII, Factor IX, Factor X and/or Factor XI is integratedinto the safe harbor locus and expressed in the cell. In certainembodiments, the safe harbor gene is a CCR5, HPRT, AAVS1, Rosa oralbumin gene. In certain embodiments, the cell is a mammalian cell. Incertain embodiments, the cell is a primate cell. In certain embodiments,the cell is a human cell. In one set of embodiments, methods forcleaving an albumin gene in a cell (e.g., a liver cell) are providedcomprising introducing, into the cell, one or more expression vectorsdisclosed herein under conditions such that the one or more proteins areexpressed and the albumin gene is cleaved. The albumin gene may bemodified, for example, by integration of a donor sequence into thecleaved albumin gene.

In certain embodiments, the method comprises genetically modifying acell to produce Factor IX, the method comprising administering to thecell the zinc finger nucleases (ZFNs) shown in Table 1 (orpolynucleotides encoding these ZFNs) and a donor encoding a Factor IXprotein. The ZFNs and donor may be on the same or different vectors inany combination, for example on 3 separate AAV vectors each carrying oneof the components; one vector carrying two of the components and aseparate vector carrying the 3^(rd) component; or one vector carryingall 3 components.

In another aspect, provided herein are methods for providing afunctional protein (e.g., Factor VIII) lacking or deficient in a mammal,or in a primate, such as a human primate, such as a human patient withhemophilia A, for example for treating hemophilia A. In some cases, thefunctional protein is a circulating plasma protein. In some cases, thefunctional protein is a non-circulating plasma protein. In some cases,the functional protein may be liver specific. In another aspect,provided herein are methods for providing a functional protein (e.g.,Factor IX) lacking or deficient in a mammal, or in a primate, such as ahuman primate, such as a human patient with hemophilia B, for examplefor treating hemophilia B. In another aspect, provided herein aremethods for providing a functional protein (e.g. Factor VII) to amammal, or in a primate, such as a human primate, such as a humanpatient, for treating hemophilia associated with Factor VII deficiency.In another aspect, provided herein are methods for providing afunctional protein (e.g. Factor X) for treating hemophilia associatedwith Factor X deficiency. In another aspect, provided herein are methodsfor providing a functional protein (e.g. Factor XI) for treatinghemophilia associated with Factor XI deficiency. In certain embodiments,the methods comprise using nucleases to integrate a sequence encoding afunctional Factor VII, Factor VIII, Factor IX, Factor X, and/or FactorXI protein in a cell in a subject in need thereof. In other embodiments,the method comprises administering a genetically modified cell(expressing a functional version of a protein that is aberrantlyexpressed in a subject with hemophilia) to the subject. Thus, anisolated cell may be introduced into the subject (ex vivo cell therapy)or a cell may be modified when it is part of the subject (in vivo). Alsoprovided is the use of the donors and/or nucleases described herein forthe treatment of a hemophilia (e.g., hemophilia A with Factor VIIIdonor, hemophilia B with Factor IX donor, Factor VII deficiency withFactor VII, Factor X deficiency with Factor X and/or Factor XIdeficiency with Factor XI), for example, in the preparation ofmedicament for treatment of hemophilia. In certain embodiments, theFactor VIII protein comprises a B-domain deletion. In certainembodiments, the Factor VIII- and/or Factor IX-encoding sequence isdelivered using a viral vector, a non-viral vector (e.g., plasmid)and/or combinations thereof.

In any of the compositions and methods described, the nuclease(s) and/ortransgene(s) may be carried on an AAV vector, including but not limitedto AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9 and AAVrh10 or pseudotypedAAV such as AAV2/8, AAV8.2, AAV2/5 and AAV2/6 and the like. In someembodiments, the AAV vector is an AAV2/6 vector. In certain embodiments,the nucleases and transgene donors are delivered using the same AAVvector types. In other embodiments, the nucleases and transgene donorsare delivered using different AAV vector types. The nucleases andtransgenes may be delivered using one or more vectors, for example, onevector carries both the transgene and nuclease(s); two vectors where onecarries the nuclease(s) (e.g., left and right ZFNs of a ZFN pair, forexample with a 2A peptide) and one carries the transgene; or threevectors where one vector carries one nuclease of a nuclease pair (e.g.,left ZFN), a separate vector carries the other nuclease of a nucleasepair (e.g., right ZFN) and a third separate vector carries thetransgene. See, FIG. 2 . In embodiments in which two or more vectors orused, the vectors may be used at the same concentrations or in differentratios, for example, the transgene donor vector(s) may be administeredat 2-fold, 3-fold, 4-fold, 5-fold or more higher concentrations than thenuclease vector(s). For example, a first nuclease, a second nucleasethat is different from the first nuclease, and a transgene donor may bedelivered in a ratio of about 1:1:1, about 1:1:2, about 1:1:3, about1:1:4, about 1:1:5, about 1:1:6, about 1:1:7, about 1:1:8, about 1:1:9,about 1:1:10, about 1:1:11, about 1:1:12, about 1:1:13, about 1:1:14,about 1:1:15, about 1:1:16, about 1:1:17, about 1:1:18, about 1:1:19,or, in some cases, about 1:1:20. In an illustrative embodiment, a firstnuclease, a second nuclease that is different from the first nuclease,and a transgene donor are delivered in a ratio of about 1:1:8. Incertain embodiments, the nucleases and/or transgene donors are deliveredvia intravenous (e.g., intra-portal vein) administration into the liverof an intact animal.

In any of the compositions and methods described herein, the proteinencoded by the transgene may comprise a Factor VIII protein, or amodified Factor VIII protein, for example a B-Domain Deleted Factor VIII(BDD-F8) or fragment thereof. In other embodiments, the protein encodedby the transgene comprises a Factor IX protein. In other embodiments,the protein encoded by the transgene comprises a Factor VII protein. Inother embodiments, the protein encoded by the transgene comprises aFactor X protein. In any of the compositions or methods describedherein, the transgene also comprises a transcriptional regulator whilein others, it does not and transcription is regulated by an endogenousregulator. In another aspect, the methods of the invention comprise acomposition for therapeutic treatment of a subject in need thereof. Insome embodiments, the composition comprises engineered stem cellscomprising a safe harbor specific nuclease, and a transgene donorencoding Factor VII, Factor VIII, Factor IX, Factor X and/or Factor XIprotein or a functional fragment and/or truncation thereof. In otherembodiments, the composition comprises engineered stem cells that havebeen modified and express a transgene donor encoding Factor VII, FactorVIII, Factor IX, Factor X, and/or Factor XI protein or a functionalfragment and/or truncation thereof.

In any of the compositions or methods described herein, the cell may bea eukaryotic cell. Non-limiting examples of suitable cells includeeukaryotic cells or cell lines such as secretory cells (e.g., livercells, mucosal cells, salivary gland cells, pituitary cells, etc.),blood cells (red blood cells), red blood precursory cells, hepaticcells, muscle cells, stem cells (e.g., embryonic stem cells, inducedpluripotent stem cells, hepatic stem cells, hematopoietic stem cells(e.g., CD34+)) or endothelial cells (e.g., vascular, glomerular, andtubular endothelial cells). Thus, the target cells may be primate cells,for example human cells, or the target cells may be mammalian cells,(including veterinary animals), for example especially nonhuman primatesand mammals of the orders Rodenta (mice, rats, hamsters), Lagomorpha(rabbits), Carnivora (cats, dogs), and Arteriodactyla (cows, pigs,sheep, goats, horses). In some aspects, the target cells comprise atissue (e.g. liver). In some aspects, the target cell is a stem cell(e.g., an embryonic stem cell, an induced pluripotent stem cell, ahepatic stem cell, etc.) or non-human animal embryo by any of themethods described herein, and then the embryo is implanted such that alive animal is born. The animal is then raised to sexual maturity andallowed to produce offspring wherein at least some of the offspringcomprise the genomic modification. The cell can also comprise an embryocell, for example, of a mouse, rat, rabbit or other mammal cell embryo.The cell may be from any organism, for example human, non-human primate,mouse, rat, rabbit, cat, dog or other mammalian cells. The cell may beisolated or may be part of an organism (e.g., subject).

In any of the methods and compositions described herein, the transgenemay be integrated into the endogenous safe harbor gene such that some,all or none of the endogenous gene is expressed, for example a fusionprotein with the integrated transgene. In some embodiments, theendogenous safe harbor gene is an albumin gene and the endogenoussequences are albumin sequences. The endogenous may be present on theamino (N)-terminal portion of the exogenous protein and/or on thecarboxy (C)-terminal portion of the exogenous protein. The albuminsequences may include full-length wild-type or mutant albumin sequencesor, alternatively, may include partial albumin amino acid sequences. Incertain embodiments, the albumin sequences (full-length or partial)serve to increase the serum half-life of the polypeptide expressed bythe transgene to which it is fused and/or as a carrier. In otherembodiments, the transgene comprises albumin sequences and is targetedfor insertion into another safe harbor within a genome. Furthermore, thetransgene may include an exogenous promoter (e.g., constitutive orinducible promoter) that drives its expression or its expression may bedriven by endogenous control sequences (e.g., endogenous albuminpromoter). In some embodiments, the donor includes additionalmodifications, including but not limited to codon optimization, additionof glycosylation sites, truncations and the like.

In some embodiments, a fusion protein comprising a zinc finger proteinand a wild-type or engineered cleavage domain or cleavage half-domainare provided.

In some embodiments, a pharmaceutical composition is providedcomprising: (i) an AAV vector comprising a polynucleotide encoding azinc finger nuclease, the zinc finger nuclease comprising a FokIcleavage domain and a zinc finger protein comprising 5 zinc fingerdomains ordered F1 to F5, wherein each zinc finger domain comprises arecognition helix region and wherein the recognition helix regions ofthe zinc finger protein are shown in the first row of Table 1 (SEQ IDNOs:4-8) (e.g., SEQ ID NO. 15); (ii) an AAV vector comprising apolynucleotide encoding a zinc finger nuclease, the zinc finger nucleasecomprising a FokI cleavage domain and a zinc finger protein comprising 6zinc finger domains ordered F1 to F6, wherein each zinc finger domaincomprises a recognition helix region and wherein the recognition helixregions of the zinc finger protein are shown in the second row of Table1 (SEQ ID NOs:9-14) (e.g., SEQ ID NO. 16); and (iii) an AAV vectorcomprising a donor encoding a Factor IX protein (e.g., SEQ ID NO. 17).

In some embodiments, (i), (ii), and (iii) are provided in a ratio about1:1:1, about 1:1:2, about 1:1:3, about 1:1:4, about 1:1:5, about 1:1:6,about 1:1:7, about 1:1:8, about 1:1:9, about 1:1:10, about 1:1:11, about1:1:12, about 1:1:13, about 1:1:14, about 1:1:15, about 1:1:16, about1:1:17, about 1:1:18, about 1:1:19, or about 1:1:20.

In some embodiments, a pharmaceutical composition is provided comprisinga first polynucleotide comprising or consisting of an amino acidsequence having 80%, 90%, 95%, 99%, 99.5% or 99.8% or more identity toSEQ ID NO. 15, a second polynucleotide comprising or consisting of anamino acid sequence having 80%, 90%, 95%, 99%, 99.5% or 99.8% or moreidentity to SEQ ID NO. 16, and, optionally, a donor sequence comprisingor consisting of an amino acid sequence having 80%, 90%, 95%, 99%, 99.5%or 99.8% or more identity to SEQ ID NO. 17.

In some embodiments, a pharmaceutical composition is provided comprisingor consisting of a first polynucleotide comprising a sequence as in SEQID NO. 15, a second polynucleotide comprising or consisting of asequence as in SEQ ID NO. 16, and, optionally, a donor sequencecomprising or consisting of a sequence as in SEQ ID NO. 17.

In any of the compositions or methods described herein, cleavage canoccur through the use of specific nucleases such as engineered zincfinger nucleases (ZFN), transcription-activator like effector nucleases(TALENs), or using the Ttago or CRISPR/Cas system with an engineeredcrRNA/tracr RNA (‘single guide RNA’) to guide specific cleavage. In someembodiments, two nickases are used to create a DSB by introducing twonicks. In some cases, the nickase is a ZFN, while in others, the nickaseis a TALEN or a CRISPR/Cas system. Targeted integration may occur viahomology directed repair mechanisms (HDR) and/or via non-homology repairmechanisms (e.g., NHEJ donor capture). The nucleases as described hereinmay bind to and/or cleave the region of interest in a coding ornon-coding region within or adjacent to the gene, such as, for example,a leader sequence, trailer sequence or intron, or within anon-transcribed region, either upstream or downstream of the codingregion. In certain embodiments, the nuclease cleaves the target sequenceat or near the binding site (e.g., the binding site shown in Table 1).Cleavage can result in modification of the gene, for example, viainsertions, deletions or combinations thereof. In certain embodiments,the modification is at or near the nuclease(s) binding and/or cleavagesite(s), for example, within 1-300 (or any value therebetween) basepairs upstream or downstream of the site(s) of cleavage, more preferablywithin 1-100 base pairs (or any value therebetween) of either side ofthe binding and/or cleavage site(s), even more preferably within 1 to 50base pairs (or any value therebetween) on either side of the bindingand/or cleavage site(s).

The methods and compositions described may be used to treat or prevent ahemophilia in a subject in need thereof. In some embodiments, a methodof treating a patient with hemophilia B is provided comprisingadministering to the patient any expression vector or pharmaceuticalcomposition described herein, wherein expression vector orpharmaceutical composition mediates targeted integration of a transgeneencoding a functional Factor IX protein into an endogenous albumin gene.In some embodiments, the compositions comprise vectors and are used totarget liver cells. In other embodiments, the compositions compriseengineered stem cells and are given to a patient as a bone marrowtransplant. In some instances, patients are partially or completelyimmunoablated prior to transplantation. In other instances, patients aretreated with one or more immunosuppressive agents before, during and/orafter nuclease-mediated modification an endogenous gene (e.g., targetedintegration of a FIX transgene into an albumin locus).

In other aspects, described herein are methods of treating and/orpreventing Hemophilia B using a system (“SB-FIX” system) comprisingengineered zinc finger nucleases (ZFNs) as shown in Table 1 tosite-specifically integrate a corrective copy of the human Factor 9(hF9) transgene into the genome of the subject's own hepatocytes invivo. Integration of the hF9 transgene is targeted to intron 1 of thealbumin locus, resulting in stable, high level, liver-specificexpression and secretion of Factor IX in to the blood. SB-FIX containsthree individual recombinant adeno-associated virus serotype 2/6(rAAV2/6) vectors, administered as a serial and consecutive intravenousdose: a first vector (SB-42906 of Table 1) encoding SBS42906, a secondvector (SB-43043) encoding SBS43043, and a third vector (SB-F9 donor,also referred to as “SB-FIX donor”) providing a DNA repair templateencoding a promoterless hF9 transgene. Delivery of all three vectorsresults in 1) specific and selective cleavage at a pre-defined site inintron 1 of the albumin locus by the pair of engineeredsequence-specific ZFNs (SBS42906 & SBS43043) and 2) stable integrationof the hF9 transgene coded by the DNA repair template at the site of theZFN-induced DNA break. Placement of the hF9 transgene into the genome,and under the control of the highly expressed endogenous albumin locusprovides permanent, liver-specific expression of Factor IX for thelifetime of the subject.

Furthermore, any of the methods described herein may further compriseadditional steps, including partial hepatectomy or treatment withsecondary agents that enhance transduction and/or induce hepatic cellsto undergo cell cycling. Examples of secondary agents include gammairradiation, UV irradiation, tritiated nucleotides such as thymidine,cis-platinum, etoposide, hydroxyurea, aphidicolin, prednisolone, carbontetrachloride and/or adenovirus.

The methods described herein can be practiced in vitro, ex vivo or invivo. In certain embodiments, the compositions are introduced into alive, intact mammal. The mammal may be at any stage of development atthe time of delivery, e.g., embryonic, fetal, neonatal, infantile,juvenile or adult. Additionally, targeted cells may be healthy ordiseased. In certain embodiments, one or more of the compositions aredelivered intravenously (e.g., to the liver via the intraportal vein,for example tail vein injection), intra-arterially, intraperitoneally,intramuscularly, into liver parenchyma (e.g., via injection), into thehepatic artery (e.g., via injection), and/or through the biliary tree(e.g., via injection).

In one particular aspect, the methods and compositions described hereininclude a therapeutic composition comprising (i) a donor transgenecoding for FVIII (ii) a nuclease (e.g., ZFN, TALENs, Ttago or CRISPR/Cassystem) targeting a locus of an endogenous gene other than FVIII,respectively, for example, targeting the endogenous albumin gene of amammal, or primate or human, such as hemophilia patient. In certainembodiments, the therapeutic composition comprises the FVIII donortransgene and the albumin gene-specific nuclease in separate,independent vectors, such as separate AAV vectors, in different amounts,which can be administered together (e.g., mixed into a single solutionor administered simultaneously) or, alternatively, which can beadministered separately (for example, administered in separate solutionswith a substantial delay, e.g., 10 minutes or more, 30 minutes or more,1 hour or more, 2 hours or more, 3 hours or more, or longer betweenrespective administrations). In certain embodiments, the therapeuticcomposition is administered to provide integration of the FVIII donortransgene into the non-FVIII locus and subsequent expression of theintegrated FVIII to achieve a therapeutic level of FVIII in the plasmaof the mammal, or primate or human, or hemophilia patient. In certainembodiments, a therapeutic level of FVIII can include, for example,greater than 2%, greater than 4%, greater than 5%, greater than 6%,greater than 8%, greater than 10%, greater than 12%, greater than 15%,greater than 20%, greater than 25%, or more of a clinically-acceptablenormal plasma concentration of FVIII. Alternatively or in addition, atherapeutic level of FVIII can include, for example, greater than 2%,greater than 4%, greater than 5%, greater than 6%, greater than 8%,greater than 10%, greater than 12%, greater than 15%, greater than 20%,greater than 25%, greater than 30%, greater than 35%, or more of theplasma concentration of functional FVIII measured in the individualmammal, or primate or human, or hemophilia patient prior toadministration of the FVIII donor transgene and the albumin genenuclease to that individual.

In another particular aspect, the methods and compositions describedherein include a therapeutic composition comprising (i) a donortransgene coding for FIX (ii) a nuclease (e.g., ZFN, TALENs, Ttago orCRISPR/Cas system) targeting a locus of an endogenous gene other thanFIX, respectively, for example, targeting the endogenous albumin gene ofa mammal, or primate or human, such as hemophilia patient. In certainembodiments, the therapeutic composition comprises the FIX donortransgene and the albumin gene nuclease in separate, independentvectors, such as separate AAV vectors, in different amounts, which canbe administered together (e.g., mixed into a single solution oradministered simultaneously) or, alternatively, which can beadministered separately ((for example, administered in separatesolutions with a substantial delay, e.g., 10 minutes or more, 30 minutesor more, 1 hour or more, 2 hours or more, 3 hours or more, or longerbetween respective administrations). In certain embodiments, thetherapeutic composition is administered to provide integration of theFIX donor transgene into the non-FIX locus and subsequent expression ofthe integrated FIX to achieve a therapeutic level of FIX in the plasmaof the mammal, or primate or human, or hemophilia patient. In certainembodiments, a therapeutic level of FIX can include, for example,greater than 2%, greater than 4%, greater than 5%, greater than 6%,greater than 8%, greater than 10%, greater than 12%, greater than 15%,greater than 20%, greater than 25%, or more of a clinically-acceptablenormal plasma concentration of FIX. Alternatively or in addition, atherapeutic level of FIX can include, for example, greater than 2%,greater than 4%, greater than 5%, greater than 6%, greater than 8%,greater than 10%, greater than 12%, greater than 15%, greater than 20%,greater than 25%, greater than 30%, greater than 35%, or more of theplasma concentration of functional FIX measured in the individualmammal, or primate or human, or hemophilia patient prior toadministration of the FIX donor transgene and the albumin gene nucleaseto that individual.

For targeting the compositions to a particular type of cell, e.g.,platelets, fibroblasts, hepatocytes, etc., one or more of theadministered compositions may be associated with a homing agent thatbinds specifically to a surface receptor of the cell. For example, thevector may be conjugated to a ligand (e.g., galactose) for which certainhepatic system cells have receptors. The conjugation may be covalent,e.g., a crosslinking agent such as glutaraldehyde, or noncovalent, e.g.,the binding of an avidinated ligand to a biotinylated vector. Anotherform of covalent conjugation is provided by engineering the AAV helperplasmid used to prepare the vector stock so that one or more of theencoded coat proteins is a hybrid of a native AAV coat protein and apeptide or protein ligand, such that the ligand is exposed on thesurface of the viral particle.

A kit, comprising the compositions (e.g., expression vectors,genetically modified cells, ZFPs, CRISPR/Cas system and/or TALENs andoptionally transgene donors) of the invention, is also provided. The kitmay comprise nucleic acids encoding the nucleases (e.g. RNA molecules ornuclease-encoding genes contained in a suitable expression vector),donor molecules, suitable host cell lines, instructions for performingthe methods of the invention, and the like.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing exemplary nucleases and donors. Shown arethree vectors: AAV2/6 Vectors for the two nucleases (SB-42906, leftZFN/ZFN 1 and SB-43043, right ZFN/ZFN2) as well as the SB-F9 donor(bottom panel).

FIG. 2 is a schematic representation showing binding of the ZFNsSBS42906 and SBS43043 to their DNA target sequences (boxed within thealbumin sequences shown in SEQ ID NO:1) at the albumin locus.

FIG. 3 is a schematic depicting nuclease-mediated homology andnon-homology directed targeted integration.

FIG. 4 is a schematic depicting targeted integration of the hF9 donor byeither NHEJ or HDR. Regardless of the mechanism by which targetedintegration occurs (NHEJ on the left and HDR on the right), the spliceacceptor sequence encoded by the donor allows for proper mRNA splicingand expression of the hF9 transgene (exons 2-8) to exon 1 from theEndogenous Albumin Promoter.

FIGS. 5A and 5B show of mRNA transcripts at the Albumin locus. FIG. 5Ais a schematic representation of alternative mRNA transcripts at thealbumin locus. Transcription at the wild type albumin locus (hALB exons1-8) yields a transcript of 2.3 kb, while after SB-FIX donor integrationthe fusion transcript hALB (exon1)-hFIX (exons 2-8) is expressed (1.7kb). The primer binding sites for RT-PCR are depicted. FIG. 5B showsanalysis of HepG2 subclones transduced either with SB-FIX donor only orwith hALB ZFNs and SB-FIX donor. The top panel shows hFIX secretion fromsubclones determined by hFIX specific ELISA after 48 hrs secretion. Themiddle panel shows the mode (HDR or NHEJ) of SB-hFIX donor integrationat the human Albumin in all subclones. The bottom panel shows RT-PCRfrom HepG2 subclones using primers, which are either specific for thewild-type human Albumin transcript or the hALB-hFIX fusion transcript.

FIGS. 6A through 6C show sequences of the hALB ZFNs and an exemplaryFactor IX donor as described herein. FIG. 6A shows the amino acidsequence of the ZFN 42906 (SEQ ID NO:15) (ZFP and cleavage domainsequence shown). FIG. 6B shows the amino acid sequence of ZFN 43043 (SEQID NO:16) (ZFP and cleavage domain sequence shown). The recognitionhelix regions of the ZFP are underlined and the FokI cleavage domainsare shown in italics (“ELD” FokI domain in ZFN 42906 and “KKR” FokIdomain in ZFN 43043). FIG. 6C (SEQ ID NO:17) shows the nucleotidesequence of an exemplary Factor IX donor. The left (base pairs 271-550)and right (base pairs 2121-2220) homology arms are shown in uppercaseand underlined. The splice acceptor sequence (base pairs 557-584) isunderlined. The codon optimized Factor IX-encoding sequence is shown inuppercase (base pairs 585-1882) and the polyadenylation signal is shownin bold (base pairs 1890-2114).

DETAILED DESCRIPTION

Disclosed herein are compositions and methods for modifying a cell toproduce one or more proteins whose expression or gene sequence, prior tomodification, is aberrant. In some embodiments, the protein isassociated with a hemophilia. The cell may be modified by targetedinsertion of a transgene encoding one or more functional proteins into asafe harbor gene (e.g., albumin) of the cell. In some embodiments, thetransgene is inserted into an endogenous albumin gene. The transgene canencode any protein or peptide involved in hemophilia, for example,Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, and/orfunctional fragments thereof. Also disclosed are methods of treating ahemophilia using a cell as described herein and/or by modifying a cell(ex vivo or in vivo) as described herein. Further described arecompositions comprising nucleic acids encoding nucleases and donormolecules for modifying a cell, and methods for modifying the cell invivo or ex vivo. Additionally, compositions comprising cells that havebeen modified by the methods and compositions of the invention aredescribed.

The genomically-modified cells described herein are typically modifiedvia nuclease-mediated (ZFN, TALEN and/or CRISPR/Cas) targetedintegration to insert a sequence encoding a therapeutic protein (e.g.,Factor VII, Factor VIII (F8), Factor IX, Factor X and/or Factor XI),wherein the protein, whose gene in an altered or aberrant state, isassociated with a hemophilia disease, into the genome of one or morecells of the subject (in vivo or ex vivo), such that the cells producethe protein in vivo. In certain embodiments, the methods furthercomprise inducing cells of the subject, particularly liver cells, toproliferate (enter the cell cycle), for example, by partial hepatectomyand/or by administration of one or more compounds that induce hepaticcells to undergo cell cycling. Subjects include but are not limited tohumans, non-human primates, veterinary animals such as cats, dogs,rabbits, rats, mice, guinea pigs, cows, pigs, horses, goats and thelike.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

The term “homology” refers to the overall relatedness between polymericmolecules, e.g., between nucleic acid molecules (e.g., DNA moleculesand/or RNA molecules) and/or between polypeptide molecules. In someembodiments, polymeric molecules are considered to be “homologous” toone another if their sequences are at least 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.8%identical. In some embodiments, polymeric molecules are considered to be“homologous” to one another if their sequences are at least 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%,99.5%, or 99.8% similar.

The term “identity” refers to the overall relatedness between polymericmolecules, e.g., between nucleic acid molecules (e.g., DNA moleculesand/or RNA molecules) and/or between polypeptide molecules. Calculationof the percent identity of two nucleic acid sequences, for example, canbe performed by aligning the two sequences for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond nucleic acid sequences for optimal alignment and non-identicalsequences can be disregarded for comparison purposes). In certainembodiments, the length of a sequence aligned for comparison purposes isat least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, 99%, 99.5%, 99.8% orsubstantially 100% of the length of the reference sequence. Thenucleotides at corresponding nucleotide positions are then compared.When a position in the first sequence is occupied by the same nucleotideas the corresponding position in the second sequence, then the moleculesare identical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using the algorithm of Meyers and Miller(CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGNprogram (version 2.0) using a PAM 120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. The percent identity between twonucleotide sequences can, alternatively, be determined using the GAPprogram in the GCG software package using an NWSgapdna.CMP matrix.Various other sequence alignment programs are available and can be usedto determine sequence identity such as, for example, Clustal.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein. See, e.g.,U.S. Pat. No. 8,586,526.

Zinc finger and TALE binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger or TALE protein. Therefore, engineered DNA bindingproteins (zinc fingers or TALEs) are proteins that are non-naturallyoccurring. Non-limiting examples of methods for engineering DNA-bindingproteins are design and selection. A designed DNA binding protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPand/or TALE designs and binding data. See, for example, U.S. Pat. Nos.6,140,081; 6,453,242; 6,534,261; and 8,586,526 see also InternationalPatent Publication Nos. WO 98/53058; WO 98/53059; WO 98/53060; WO02/016536; and WO 03/016496.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; and 6,200,759;International Patent Publication Nos. WO 95/19431; WO 96/06166; WO98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; and WO02/099084.

“TtAgo” is a prokaryotic Argonaute protein thought to be involved ingene silencing. TtAgo is derived from the bacteria Thermus thermophilus.See, e.g., Swarts et al., ibid, G. Sheng et al. (2013) Proc. Natl. Acad.Sci. U.S.A. 111:652). A “TtAgo system” is all the components requiredincluding, for example, guide DNAs for cleavage by a TtAgo enzyme.

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides, including but not limited to, donor captureby non-homologous end joining (NHEJ) and homologous recombination. Forthe purposes of this disclosure, “homologous recombination (HR)” refersto the specialized form of such exchange that takes place, for example,during repair of double-strand breaks in cells via homology-directedrepair mechanisms. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the one that experienced the double-strand break), and is variouslyknown as “non-crossover gene conversion” or “short tract geneconversion,” because it leads to the transfer of genetic informationfrom the donor to the target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or “synthesis-dependent strand annealing,” in which the donor isused to resynthesize genetic information that will become part of thetarget, and/or related processes. Such specialized HR often results inan alteration of the sequence of the target molecule such that part orall of the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide.

In the methods of the disclosure, one or more targeted nucleases asdescribed herein can create a double-stranded break in the targetsequence (e.g., cellular chromatin) at a predetermined site, and a“donor” polynucleotide, having homology to the nucleotide sequence inthe region of the break, can be introduced into the cell. The presenceof the double-stranded break has been shown to facilitate integration ofthe donor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. The use of the terms “replace” or“replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another.

In any of the methods described herein, additional pairs of zinc-fingerproteins or TALEN can be used for additional double-stranded cleavage ofadditional target sites within the cell.

In certain embodiments of methods for targeted recombination and/orreplacement and/or alteration of a sequence in a region of interest incellular chromatin, a chromosomal sequence is altered by homologousrecombination with an exogenous “donor” nucleotide sequence. Suchhomologous recombination is stimulated by the presence of adouble-stranded break in cellular chromatin, if sequences homologous tothe region of the break are present.

In any of the methods described herein, the first nucleotide sequence(the “donor sequence”) can contain sequences that are homologous, butnot identical, to genomic sequences in the region of interest, therebystimulating homologous recombination to insert a non-identical sequencein the region of interest. Thus, in certain embodiments, portions of thedonor sequence that are homologous to sequences in the region ofinterest exhibit between about 80 to 99% (or any integer therebetween)sequence identity to the genomic sequence that is replaced. In otherembodiments, the homology between the donor and genomic sequence ishigher than 99%, for example if only 1 nucleotide differs as betweendonor and genomic sequences of over 100 contiguous base pairs. Incertain cases, a non-homologous portion of the donor sequence cancontain sequences not present in the region of interest, such that newsequences are introduced into the region of interest. In theseinstances, the non-homologous sequence is generally flanked by sequencesof 50-1,000 base pairs (or any integral value therebetween) or anynumber of base pairs greater than 1,000, that are homologous oridentical to sequences in the region of interest. In other embodiments,the donor sequence is non-homologous to the first sequence, and isinserted into the genome by non-homologous recombination mechanisms.

Any of the methods described herein can be used for partial or completeinactivation of one or more target sequences in a cell by targetedintegration of donor sequence that disrupts expression of the gene(s) ofinterest. Cell lines with partially or completely inactivated genes arealso provided.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or noncoding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence may produce one or more RNA molecules(e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs(miRNAs), etc.).

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Pat. Nos. 7,914,796; 8,034,598; and 8,623,618, incorporated hereinby reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome” is a chromatin complex comprising all or a portion of thegenome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogenous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequence may beintroduced into a cell line originally derived from a mouse or hamster.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPor TALE DNA-binding domain and one or more activation domains) andfusion nucleic acids (for example, a nucleic acid encoding the fusionprotein described supra). Examples of the second type of fusion moleculeinclude, but are not limited to, a fusion between a triplex-formingnucleic acid and a polypeptide, and a fusion between a minor groovebinder and a nucleic acid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, random mutation) can be used to modulateexpression. Gene inactivation refers to any reduction in gene expressionas compared to a cell that does not include a ZFP as described herein.Thus, gene inactivation may be partial or complete.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFPDNA-binding domain is fused to an activation domain, the ZFP DNA-bindingdomain and the activation domain are in operative linkage if, in thefusion polypeptide, the ZFP DNA-binding domain portion is able to bindits target site and/or its binding site, while the activation domain isable to upregulate gene expression. When a fusion polypeptide in which aZFP DNA-binding domain is fused to a cleavage domain, the ZFPDNA-binding domain and the cleavage domain are in operative linkage if,in the fusion polypeptide, the ZFP DNA-binding domain portion is able tobind its target site and/or its binding site, while the cleavage domainis able to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and International Patent Publication No. WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “safe harbor” locus is a locus within the genome wherein a gene may beinserted without any deleterious effects on the host cell. Mostbeneficial is a safe harbor locus in which expression of the insertedgene sequence is not perturbed by any read-through expression fromneighboring genes. Non-limiting examples of safe harbor loci that aretargeted by nuclease(s) include CCR5, CCR5, HPRT, AAVS1, Rosa andalbumin. See, e.g., U.S. Pat. Nos. 7,951,925 and 8,110,379; U.S. PatentPublication Nos. 2008/0159996; 2010/0218264; 2012/0017290; 2011/0265198;2013/0137104; 2013/0122591; 2013/0177983; and 2013/0177960 and U.S.Provisional Application No. 61/823,689.

Nucleases

Described herein are compositions comprising nucleases, such asnucleases that are useful in integration of a sequence encoding afunctional clotting factor (e.g., Factor VIII and/or Factor IX) proteinin the genome of a cell from or in a subject with hemophilia A or B. Incertain embodiments, the nuclease is naturally occurring. In otherembodiments, the nuclease is non-naturally occurring, i.e., engineeredin the DNA-binding domain and/or cleavage domain. For example, theDNA-binding domain of a naturally-occurring nuclease may be altered tobind to a selected target site (e.g., a meganuclease that has beenengineered to bind to site different than the cognate binding site). Inother embodiments, the nuclease comprises heterologous DNA-binding andcleavage domains (e.g., zinc finger nucleases; TAL-effector domain DNAbinding proteins; meganuclease DNA-binding domains with heterologouscleavage domains) and/or a CRISPR/Cas system utilizing an engineeredsingle guide RNA).

A. DNA-Binding Domains

Any DNA-binding domain can be used in the compositions and methodsdisclosed herein, including but not limited to a zinc finger DNA-bindingdomain, a TALE DNA binding domain, the DNA-binding portion of aCRISPR/Cas nuclease, or a DNA-binding domain from a meganuclease.

In certain embodiments, the nuclease is a naturally occurring orengineered (non-naturally occurring) meganuclease (homing endonuclease).Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce,I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI,I-TevII and I-TevIII. Their recognition sequences are known. See alsoU.S. Pat. Nos. 5,420,032; 6,833,252; Belfort et al. (1997) Nucleic AcidsRes. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al.(1994) Nucleic Acids Res. 22:1125-1127; Jasin (1996) Trends Genet.12:224-228; Gimble et al. (1996)J Mol. Biol. 263:163-180; Argast et al.(1998)J Mol. Biol. 280:345-353 and the New England Biolabs catalogue.Engineered meganucleases are described for example in U.S. PatentPublication No. 2007/0117128. The DNA-binding domains of the homingendonucleases and meganucleases may be altered in the context of thenuclease as a whole (i.e., such that the nuclease includes the cognatecleavage domain) or may be fused to a heterologous cleavage domain.DNA-binding domains from meganucleases may also exhibit nucleaseactivity (e.g., cTALENs).

In other embodiments, the DNA-binding domain comprises a naturallyoccurring or engineered (non-naturally occurring) TAL effector DNAbinding domain. See, e.g., U.S. Pat. No. 8,586,526, incorporated byreference in its entirety herein. The plant pathogenic bacteria of thegenus Xanthomonas are known to cause many diseases in important cropplants. Pathogenicity of Xanthomonas depends on a conserved type IIIsecretion (T3 S) system which injects more than 25 different effectorproteins into the plant cell. Among these injected proteins aretranscription activator-like (TAL) effectors which mimic planttranscriptional activators and manipulate the plant transcriptome (seeKay et al. (2007) Science 318:648-651). These proteins contain a DNAbinding domain and a transcriptional activation domain. One of the mostwell characterized TAL-effectors is AvrBs3 from Xanthomonas campestgrispv. Vesicatoria (see Bonas et al. (1989) Mol Gen Genet 218:127-136 andInternational Patent Publication No. WO 2010079430). TAL-effectorscontain a centralized domain of tandem repeats, each repeat containingapproximately 34 amino acids, which are key to the DNA bindingspecificity of these proteins. In addition, they contain a nuclearlocalization sequence and an acidic transcriptional activation domain(for a review see Schornack et al. (2006) J Plant Physiol163(3):256-272). In addition, in the phytopathogenic bacteria Ralstoniasolanacearum two genes, designated brg11 and hpx17 have been found thatare homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar 1 strain GMI1000 and in the biovar 4 strain RS1000(See Heuer et al. (2007) Appl and Envir Micro 73(13):4379-4384). Thesegenes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 base pairs in the repeat domain of hpx17.However, both gene products have less than 40% sequence identity withAvrBs3 family proteins of Xanthomonas. See, e.g., U.S. Pat. No.8,586,526, incorporated by reference in its entirety herein.

Specificity of these TAL effectors depends on the sequences found in thetandem repeats. The repeated sequence comprises approximately 102 basepair and the repeats are typically 91-100% homologous with each other(Bonas et al., ibid). Polymorphism of the repeats is usually located atpositions 12 and 13 and there appears to be a one-to-one correspondencebetween the identity of the hypervariable diresidues (the repeatvariable diresidue or RVD region) at positions 12 and 13 with theidentity of the contiguous nucleotides in the TAL-effector's targetsequence (see Moscou and Bogdanove (2009) Science 326:1501 and Boch etal. (2009) Science 326:1509-1512). Experimentally, the natural code forDNA recognition of these TAL-effectors has been determined such that anHD sequence at positions 12 and 13 (Repeat Variable Diresidue or RVD)leads to a binding to cytosine (C), NG binds to T, NI to A, C, G or T,NN binds to A or G, and ING binds to T. These DNA binding repeats havebeen assembled into proteins with new combinations and numbers ofrepeats, to make artificial transcription factors that are able tointeract with new sequences and activate the expression of anon-endogenous reporter gene in plant cells (Boch et al., ibid).Engineered TAL proteins have been linked to a FokI cleavage half domainto yield a TAL effector domain nuclease fusion (TALEN), including TALENswith atypical RVDs. See, e.g., U.S. Pat. No. 8,586,526.

In some embodiments, the TALEN comprises a endonuclease (e.g., FokI)cleavage domain or cleavage half-domain. In other embodiments, theTALE-nuclease is a mega TAL. These mega TAL nucleases are fusionproteins comprising a TALE DNA binding domain and a meganucleasecleavage domain. The meganuclease cleavage domain is active as a monomerand does not require dimerization for activity. (See Boissel et al.(2013) Nucl Acid Res 42(4):2591-601, doi: 10.1093/nar/gkt1224).

In still further embodiments, the nuclease comprises a compact TALEN.These are single chain fusion proteins linking a TALE DNA binding domainto a TevI nuclease domain. The fusion protein can act as either anickase localized by the TALE region, or can create a double strandbreak, depending upon where the TALE DNA binding domain is located withrespect to the TevI nuclease domain (see Beurdeley et al. (2013) NatComm 4:1762:1-8 DOI: 10.1038/ncomms2782). In addition, the nucleasedomain may also exhibit DNA-binding functionality. Any TALENs may beused in combination with additional TALENs (e.g., one or more TALENs(cTALENs or FokI-TALENs) with one or more mega-TALEs.

In certain embodiments, the DNA binding domain comprises a zinc fingerprotein. Preferably, the zinc finger protein is non-naturally occurringin that it is engineered to bind to a target site of choice. See, forexample, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al.(2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) NatureBiotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol.12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416;U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558;7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; and7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528;and 2005/0267061, all incorporated herein by reference in theirentireties.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as International Patent Publication Nos. WO 98/37186; WO 98/53057;WO 00/27878; and WO 01/88197 and GB 2,338,237. In addition, enhancementof binding specificity for zinc finger binding domains has beendescribed, for example, in co-owned International Patent Publication No.WO 02/077227.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. Pat. Nos.6,140,081; 5,789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988;6,013,453; and 6,200,759; International Patent Publication Nos. WO95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO01/60970; WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO98/53060; WO 02/016536; and WO 03/016496.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

B. Cleavage Domains

Any suitable cleavage domain can be operatively linked to a DNA-bindingdomain to form a nuclease. such as a zinc finger nuclease, a TALEN, or aCRISPR/Cas nuclease system. See, e.g., U.S. Pat. Nos. 7,951,925;8,110,379 and 8,586,526; U.S. Patent Publication Nos. 2008/0159996;2010/00218264; 2012/0017290; 2011/0265198; 2013/0137104; 2013/0122591;2013/0177983; and 2013/0177960 and U.S. Provisional Patent ApplicationNo. 61/823,689.

As noted above, the cleavage domain may be heterologous to theDNA-binding domain, for example a zinc finger DNA-binding domain and acleavage domain from a nuclease or a TALEN DNA-binding domain and acleavage domain, or meganuclease DNA-binding domain and cleavage domainfrom a different nuclease. Heterologous cleavage domains can be obtainedfrom any endonuclease or exonuclease. Exemplary endonucleases from whicha cleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., 51 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any number of nucleotides or nucleotide pairs canintervene between two target sites (e.g., from 2 to 50 nucleotide pairsor more). In general, the site of cleavage lies between the targetsites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme FokI catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150; and 5,487,994; as wellas Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is FokI. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95:10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the FokI enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-FokI fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and twoFokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-FokI fusionsare provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPatent Publication No. WO 07/014275, incorporated herein in itsentirety. Additional restriction enzymes also contain separable bindingand cleavage domains, and these are contemplated by the presentdisclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res.31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 2005/0064474; 2006/0188987;2007/0305346; and 2008/0131962, the disclosures of all of which areincorporated by reference in their entireties herein. Amino acidresidues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496,498, 499, 500, 531, 534, 537, and 538 of FokI are all targets forinfluencing dimerization of the FokI cleavage half-domains.

Exemplary engineered cleavage half-domains of FokI that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFokI and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Pat. Nos. 7,914,796 and 8,034,598, the disclosures of which areincorporated by reference in their entireties for all purposes. Incertain embodiments, the engineered cleavage half-domain comprisesmutations at positions 486, 499 and 496 (numbered relative to wild-typeFokI), for instance mutations that replace the wild type Gln (Q) residueat position 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). (See U.S. Pat. No. 8,623,618). In otherembodiments, the engineered cleavage half domain comprises the “Sharkey”and/or “Sharkey mutations” (see Guo et al. (2010) J. Mol. Biol.400(1):96-107).

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (FokI) as described in U.S. Pat. Nos.7,888,121; 7,914,796; 8,034,598; and 8,623,618.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see, e.g. U.S.Patent Publication No. 2009/0068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in International PatentPublication No. WO 2009/042163 and U.S. Patent Publication No.2009/0068164. Nuclease expression constructs can be readily designedusing methods known in the art. See, e.g., U.S. Patent Publication Nos.2003/0232410; 2005/0208489; 2005/0026157; 2005/0064474; 2006/0188987;2006/0063231; and International Patent Publication No. WO 07/014275.Expression of the nuclease may be under the control of a constitutivepromoter or an inducible promoter, for example the galactokinasepromoter which is activated (de-repressed) in the presence of raffinoseand/or galactose and repressed in presence of glucose.

In certain embodiments, the nuclease comprises a CRISPR/Cas system. TheCRISPR (clustered regularly interspaced short palindromic repeats)locus, which encodes RNA components of the system, and the cas(CRISPR-associated) locus, which encodes proteins (Jansen et al. (2002)Mol. Microbiol. 43:1565-1575; Makarova et al. (2002) Nucleic Acids Res.30:482-496; Makarova et al. (2006) Biol. Direct 1:7; Haft et al. (2005)PLoS Comput. Biol. 1:e60) make up the gene sequences of the CRISPR/Casnuclease system. CRISPR loci in microbial hosts contain a combination ofCRISPR-associated (Cas) genes as well as non-coding RNA elements capableof programming the specificity of the CRISPR-mediated nucleic acidcleavage.

The Type II CRISPR is one of the most well characterized systems andcarries out targeted DNA double-strand break in four sequential steps.First, two non-coding RNA, the pre-crRNA array and tracrRNA, aretranscribed from the CRISPR locus. Second, tracrRNA hybridizes to therepeat regions of the pre-crRNA and mediates the processing of pre-crRNAinto mature crRNAs containing individual spacer sequences. Third, themature crRNA:tracrRNA complex directs Cas9 to the target DNA viaWatson-Crick base-pairing between the spacer on the crRNA and theprotospacer on the target DNA next to the protospacer adjacent motif(PAM), an additional requirement for target recognition. Finally, Cas9mediates cleavage of target DNA to create a double-stranded break withinthe protospacer. Activity of the CRISPR/Cas system comprises of threesteps: (i) insertion of alien DNA sequences into the CRISPR array toprevent future attacks, in a process called ‘adaptation’, (ii)expression of the relevant proteins, as well as expression andprocessing of the array, followed by (iii) RNA-mediated interferencewith the alien nucleic acid. Thus, in the bacterial cell, several of theso-called ‘Cas’ proteins are involved with the natural function of theCRISPR/Cas system and serve roles in functions such as insertion of thealien DNA etc.

In certain embodiments, Cas protein may be a “functional derivative” ofa naturally occurring Cas protein. A “functional derivative” of a nativesequence polypeptide is a compound having a qualitative biologicalproperty in common with a native sequence polypeptide. “Functionalderivatives” include, but are not limited to, fragments of a nativesequence and derivatives of a native sequence polypeptide and itsfragments, provided that they have a biological activity in common witha corresponding native sequence polypeptide. A biological activitycontemplated herein is the ability of the functional derivative tohydrolyze a DNA substrate into fragments. The term “derivative”encompasses both amino acid sequence variants of polypeptide, covalentmodifications, and fusions thereof. Suitable derivatives of a Caspolypeptide or a fragment thereof include but are not limited tomutants, fusions, covalent modifications of Cas protein or a fragmentthereof. Cas protein, which includes Cas protein or a fragment thereof,as well as derivatives of Cas protein or a fragment thereof, may beobtainable from a cell or synthesized chemically or by a combination ofthese two procedures. The cell may be a cell that naturally produces Casprotein, or a cell that naturally produces Cas protein and isgenetically engineered to produce the endogenous Cas protein at a higherexpression level or to produce a Cas protein from an exogenouslyintroduced nucleic acid, which nucleic acid encodes a Cas that is sameor different from the endogenous Cas. In some case, the cell does notnaturally produce Cas protein and is genetically engineered to produce aCas protein.

Exemplary CRISPR/Cas nuclease systems targeted to safe harbor and othergenes are disclosed for example, in U.S. Provisional Patent ApplicationNo. 61/823,689.

Thus, the nuclease comprises a DNA-binding domain in that specificallybinds to a target site in any gene into which it is desired to insert adonor (transgene).

Target Sites

As described in detail above, DNA-binding domains can be engineered tobind to any sequence of choice, for example in a safe-harbor locus suchas albumin. An engineered DNA-binding domain can have a novel bindingspecificity, compared to a naturally-occurring DNA-binding domain.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.Rational design of TAL-effector domains can also be performed. See,e.g., U.S. Pat. No. 8,586,526.

Exemplary selection methods applicable to DNA-binding domains, includingphage display and two-hybrid systems, are disclosed in U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466;6,200,759; and 6,242,568; as well as International Patent PublicationNos. WO 98/37186; WO 98/53057; WO 00/27878; and WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-ownedInternational Patent Publication No. WO 02/077227.

Selection of target sites; nucleases and methods for design andconstruction of fusion proteins (and polynucleotides encoding same) areknown to those of skill in the art and described in detail in U.S.Patent Publication Nos. 2005/0064474 and 2006/0188987, incorporated byreference in their entireties herein.

In addition, as disclosed in these and other references, DNA-bindingdomains (e.g., multi-fingered zinc finger proteins) may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids. See, e.g., U.S. Pat. Nos. 6,479,626;6,903,185; and 7,153,949 for exemplary linker sequences 6 or more aminoacids in length. The proteins described herein may include anycombination of suitable linkers between the individual DNA-bindingdomains of the protein. See, also, U.S. Pat. No. 8,586,526.

For treatment of hemophilia via targeted insertion of a sequenceencoding a functional Factor VIII and/or Factor IX protein, any desiredsite of insertion in the genome of the subject is cleaved with anuclease, which stimulates targeted insertion of the donorpolynucleotide carrying the Factor VIII- and/or Factor IX-encodingsequence. DNA-binding domains of the nucleases may be targeted to anydesired site in the genome. In certain embodiments, the DNA-bindingdomain of the nuclease is targeted to an endogenous safe harbor locus,for example an endogenous albumin locus.

In certain embodiments, a pair of dimerizing zinc finger nucleases asshown in FIGS. 6A and 6B are used for cleavage of an albumin gene tofacilitate targeted integration of a Factor IX donor. The ZFNs comprisea DNA-binding domain having the recognition helix regions shown in Table1 and an engineered FokI cleavage domain such that the ZFNs form anobligate heterodimer (e.g., one member of the pair includes an “ELD”FokI domain and the other member of the pair includes a “KKR” FokIdomain.

ZFNs having homology or identity to the ZFNs described herein are alsoprovided, including but not limited to ZFN sequences having at least25%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%sequence homology or identity. Furthermore, it will be understood thatthe homology is typically determined for residues outside therecognition helix regions, including but not limited to the FokI domain,the linker domain, the zinc finger backbone residues (e.g., any residuesoutside the recognition helix region). As any zinc finger backbone(context) and/or cleavage domain can be used, significant variation inhomology outside the recognition helix regions zinc finger residues canbe present.

Donor Sequences

For treating hemophilia, the donor sequence (also called an “exogenoussequence” or “donor” or “transgene”) comprises a sequence encoding afunctional clotting factor protein, or part thereof, to result in asequence encoding and expressing a functional clotting factor proteinfollowing donor integration. Non-limiting examples of clotting factorprotein transgenes include Factor VIII and/or Factor IX, includingfunctional fragments of these proteins. In certain embodiments, theB-domain of the Factor VIII protein is deleted. See, e.g., Chuah et al.(2003) Blood 101(5):1734-1743. In other embodiments, the transgenecomprises a sequence encoding a functional Factor IX protein, or partthereof, to result in a sequence encoding and expressing a functionFactor IX protein following donor integration.

The donor molecule may be inserted into an endogenous gene such thatall, some or none of the endogenous gene is expressed. For example, atransgene comprising functional clotting factor protein (e.g., FactorVIII and/or Factor IX) sequences as described herein may be insertedinto an endogenous albumin locus such that some or none of theendogenous albumin is expressed with the transgene.

The donor (transgene) sequence can be introduced into the cell prior to,concurrently with, or subsequent to, expression of the fusion protein(s)(e.g., nucleases). The donor polynucleotide may contain sufficienthomology (continuous or discontinuous regions) to a genomic sequence tosupport homologous recombination (or homology-directed repair) betweenit and the genomic sequence to which it bears homology or,alternatively, donor sequences can be integrated via non-HDR mechanisms(e.g., NHEJ donor capture), in which case the donor polynucleotide(e.g., vector) need not containing sequences that are homologous to theregion of interest in cellular chromatin. See, e.g., U.S. Pat. Nos.7,888,121 and 7,972,843 and U.S. Patent Publication Nos. 2011/0281361;2010/0047805; and 2011/0207221.

The donor polynucleotide can be DNA or RNA, single-stranded,double-stranded or partially single- and partially double-stranded andcan be introduced into a cell in linear or circular (e.g., minicircle)form. See, e.g., U.S. Patent Publication Nos. 2010/0047805;2011/0281361; and 2011/0207221. If introduced in linear form, the endsof the donor sequence can be protected (e.g., from exonucleolyticdegradation) by methods known to those of skill in the art. For example,one or more dideoxynucleotide residues are added to the 3′ terminus of alinear molecule and/or self-complementary oligonucleotides are ligatedto one or both ends. See, for example, Chang et al. (1987) Proc. Natl.Acad. Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889.Additional methods for protecting exogenous polynucleotides fromdegradation include, but are not limited to, addition of terminal aminogroup(s) and the use of modified internucleotide linkages such as, forexample, phosphorothioates, phosphoramidates, and O-methyl ribose ordeoxyribose residues. A polynucleotide can be introduced into a cell aspart of a vector molecule having additional sequences such as, forexample, replication origins, promoters and genes encoding antibioticresistance. Moreover, donor polynucleotides can be introduced as nakednucleic acid, as nucleic acid complexed with an agent such as a liposomeor poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV,herpesvirus, retrovirus, lentivirus).

In some embodiments, the donor may be inserted so that its expression isdriven by the endogenous promoter at the integration site (e.g., theendogenous albumin promoter when the donor is integrated into thepatient's albumin locus). In such cases, the transgene may lack controlelements (e.g., promoter and/or enhancer) that drive its expression(e.g., also referred to as a “promoterless construct”). Nonetheless, itwill be apparent that in other cases the donor may comprise a promoterand/or enhancer, for example a constitutive promoter or an inducible ortissue specific (e.g., liver- or platelet-specific) promoter that drivesexpression of the functional protein upon integration. The donorsequence can be integrated specifically into any target site of choice.

When albumin sequences (endogenous or part of the transgene) areexpressed with the transgene, the albumin sequences may be full-lengthsequences (wild-type or mutant) or partial sequences. Preferably thealbumin sequences are functional. Non-limiting examples of the functionof these full length or partial albumin sequences include increasing theserum half-life of the polypeptide expressed by the transgene (e.g.,therapeutic gene) and/or acting as a carrier.

Furthermore, although not required for expression, exogenous sequencesmay also include transcriptional or translational regulatory sequences,for example, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.

Any of the donor sequences may include one or more of the followingmodifications: codon optimization (e.g., to human codons) and/oraddition of one or more glycosylation sites. See, e.g., McIntosh et al.(2013) Blood (17):3335-44.

Delivery

The nucleases, polynucleotides encoding these nucleases, donorpolynucleotides and compositions comprising the proteins and/orpolynucleotides described herein may be delivered in vivo or ex vivo byany suitable means.

Methods of delivering nucleases as described herein are described, forexample, in U.S. Pat. Nos. 8,586,526; 6,453,242; 6,503,717; 6,534,261;6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539;7,013,219; and 7,163,824, the disclosures of all of which areincorporated by reference herein in their entireties. Nucleases and/ordonor constructs as described herein may also be delivered using vectorscontaining sequences encoding one or more of the zinc finger protein(s),TALEN protein(s) and/or a CRISPR/Cas system. Any vector systems may beused including, but not limited to, plasmid vectors, retroviral vectors,lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirusvectors and adeno-associated virus vectors, etc. See, also, U.S. Pat.Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219;and 7,163,824, incorporated by reference herein in their entireties.Furthermore, it will be apparent that any of these vectors may compriseone or more of the sequences needed. Thus, when one or more nucleasesand a donor construct are introduced into the cell, the nucleases and/ordonor polynucleotide may be carried on the same vector or on differentvectors. When multiple vectors are used, each vector may comprise asequence encoding one or multiple nucleases and/or donor constructs. Incertain embodiments, one vector is used to carry both the transgene andnuclease(s). In other embodiments, two vector are used (the same ordifferent vector types), where one vector carries the nuclease(s) (e.g.,left and right ZFNs of a ZFN pair, for example with a 2A peptide) andone carries the transgene. In still further embodiments, three vectorsare used where the first vector carries one nuclease of a nuclease pair(e.g., left ZFN), the second vector carries the other nuclease of anuclease pair (e.g., right ZFN) and the third vector carries thetransgene. See, FIG. 2 .

The donors and/or nuclease may be used at any suitable concentrations.In certain embodiments, the donor and separate nuclease vector(s) areused the same concentration. In other embodiments, the donor andseparate nuclease vector(s) are used at different concentrations, forexample, 2-, 3-, 4-, 5-, 10-, 15-, 20- or more fold of one vector thanother (e.g., more donor vector(s) than nuclease vector(s). For example,a nuclease vector (or nuclease vectors) and a donor vector may be usedin a ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5,about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11,about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17,about 1:18, about 1:19, or, in some cases, about 1:20. In someembodiments, a first nuclease vector, a second nuclease vector that isdifferent from the first nuclease vector, and a donor vector may be usedin a ratio of about 1:1:1, about 1:1:2, about 1:1:3, about 1:1:4, about1:1:5, about 1:1:6, about 1:1:7, about 1:1:8, about 1:1:9, about 1:1:10,about 1:1:11, about 1:1:12, about 1:1:13, about 1:1:14, about 1:1:15,about 1:1:16, about 1:1:17, about 1:1:18, about 1:1:19, or, in somecases, about 1:1:20. In an illustrative embodiment, a first nucleasevector, a second nuclease vector that is different from the firstnuclease, and a donor vector are used in a ratio of about 1:1:8. WhenAAV vectors are used for delivery, for example, the donor- and/ornuclease-comprising viral vector(s) may be between about 1×10⁸ and about1×10¹⁵ vector genomes per kg per dose (e.g., cell or animal).

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding nucleases and donor constructs incells (e.g., mammalian cells) and target tissues. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome or poloxamer.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of in vivo delivery of engineered DNA-binding proteins and fusionproteins comprising these binding proteins, see, e.g., Rebar (2004)Expert Opinion Invest. Drugs 13(7):829-839; Rossi et al. (2007) NatureBiotech. 25(12):1444-1454 as well as general gene delivery referencessuch as Anderson (1992) Science 256:808-813; Nabel & Felgner (1993)TIBTECH 11:211-217; Mitani & Caskey (1993) TIBTECH 11:162-166); Dillon(1993) TIBTECH 11:167-175; Miller (1992) Nature 357:455-460; Van Brunt(1988) Biotechnology 6(10):1149-1154; Vigne (1995) Restorative Neurologyand Neuroscience 8:35-36; Kremer & Perricaudet (1995) British MedicalBulletin 51(1):31-44; Haddada et al., in Current Topics in Microbiologyand Immunology Doerfler and Bohm (eds.) (1995); and Yu et al. (1994)Gene Therapy 1:13-26.

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Sonoporationusing, e.g., the Sonitron 2000 system (Rich-Mar) can also be used fordelivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by AmaxaBiosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc., (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, International PatentPublication Nos. WO 91/17424, WO 91/16024.

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal (1995) Science 270:404-410; Blaese et al.(1995) Cancer Gene Ther. 2:291-297; Behr et al. (1994) BioconjugateChem. 5:382-389; Remy et al. (1994) Bioconjugate Chem. 5:647-654; Gao etal. (1995) Gene Therapy 2:710-722; Ahmad et al. (1992) Cancer Res.52:4817-4820; U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975;4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet al. (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered nucleases and/or donors take advantage ofhighly evolved processes for targeting a virus to specific cells in thebody and trafficking the viral payload to the nucleus. Viral vectors canbe administered directly to patients (in vivo) or they can be used totreat cells in vitro and the modified cells are administered to patients(ex vivo). Conventional viral based systems for the delivery ofnucleases and/or donors include, but are not limited to, retroviral,lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplexvirus vectors for gene transfer. Integration in the host genome ispossible with the retrovirus, lentivirus, and adeno-associated virusgene transfer methods, often resulting in long term expression of theinserted transgene. Additionally, high transduction efficiencies havebeen observed in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al. (1992) J. Virol.66:2731-2739; Johann et al. (1992) J Virol. 66:1635-1640; Sommerfelt etal. (1990) Virol. 176:58-59; Wilson et al. (1989) J. Virol.63:2374-2378; Miller et al. (1991) J Virol. 65:2220-2224; InternationalPatent Publication No. WO 94/26877 (PCT/US94/05700)).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al. (1987) Virology160:38-47; U.S. Pat. No. 4,797,368; International Patent Publication No.WO 93/24641; Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994)J. Clin. Invest. 94:1351. Construction of recombinant AAV vectors isdescribed in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al. (1985)Mol. Cell. Biol. 5:3251-3260;Tratschin et al. (1984)Mol. Cell. Biol. 4:2072-2081; Hermonat & Muzyczka(1984) PNAS 81:6466-6470; and Samulski et al. (1989) J. Virol.63:3822-3828.

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al. (1995) Blood 85:3048-305; Kohn et al.(1995) Nat. Med. 1:1017-102; Malech et al. (1997) PNAS 94:2212133-12138). PA317/pLASN was the first therapeutic vector used in agene therapy trial. (Blaese et al. (1995) Science 270:475-480).Transduction efficiencies of 50% or greater have been observed for MFG-Spackaged vectors. (Ellem et al. (1997) Immunol Immunother. 44(1):10-20;Dranoff et al. (1997) Hum. Gene Ther. 1:111-2.

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 base pair invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al. (1998) Lancet 351(9117):1702-3; Kearns et al. (1996) GeneTher. 9:748-55). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5,AAV6, AAV8, AAV9 and AAVrh10 or pseudotyped AAV such as AAV2/8, AAV8.2,AAV2/5 and AAV2/6 and any novel AAV serotype can also be used inaccordance with the present invention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al. (1998) Hum.Gene Ther. 7:1083-9). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.(1996) Infection 24(1):5-10; Sterman et al. (1998) Hum. Gene Ther.9(7):1083-1089; Welsh et al. (1995) Hum. Gene Ther. 2:205-18; Alvarez etal. (1997) Hum. Gene Ther. 5:597-613; Topf et al. (1998) Gene Ther.5:507-513; Sterman et al. (1998) Hum. Gene Ther. 7:1083-1089.

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al. (1995) Proc. Natl.Acad. Sci. USA 92:9747-9751, reported that Moloney murine leukemia viruscan be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingnucleases and/or donor constructs can also be administered directly toan organism for transduction of cells in vivo. Alternatively, naked DNAcan be administered. Administration is by any of the routes normallyused for introducing a molecule into ultimate contact with blood ortissue cells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Vectors suitable for introduction of polynucleotides (e.g.nuclease-encoding and/or Factor VII, Factor VIII, FIX, Factor X and/orFactor XI-encoding) described herein include non-integrating lentivirusvectors (IDLV). See, for example, Ory et al. (1996) Proc. Natl. Acad.Sci. USA 93:11382-11388; Dull et al. (1998) J. Virol. 72:8463-8471;Zuffery et al. (1998) J. Virol. 72:9873-9880; Follenzi et al. (2000)Nature Genetics 25:217-222; U.S. Patent Publication No. 2009/054985.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

It will be apparent that the nuclease-encoding sequences and donorconstructs can be delivered using the same or different systems. Forexample, the nucleases and donors can be carried by the same vector(e.g., AAV). Alternatively, a donor polynucleotide can be carried by aplasmid, while the one or more nucleases can be carried by a differentvector (e.g., AAV vector). Furthermore, the different vectors can beadministered by the same or different routes (intramuscular injection,tail vein injection, other intravenous injection, intraperitonealadministration and/or intramuscular injection. The vectors can bedelivered simultaneously or in any sequential order.

Thus, in certain embodiments, the instant disclosure includes in vivo orex vivo treatment of Hemophilia A, via nuclease-mediated integration ofFactor VIII-encoding sequence. The disclosure also includes in vivo orex vivo treatment of Hemophilia B, via nuclease-mediated integration ofa Factor IX encoding sequence. Similarly, the disclosure includes thetreatment of Factor VII deficiency, Factor X, or Factor XI deficiencyrelated hemophilias via nuclease-mediated integration of a Factor VII,Factor X, or Factor XI encoding sequence, respectively. The compositionsare administered to a human patient in an amount effective to obtain thedesired concentration of the therapeutic Factor VII, Factor VIII, FactorIX or Factor X polypeptide in the serum, the liver or the target cells.Administration can be by any means in which the polynucleotides aredelivered to the desired target cells. For example, both in vivo and exvivo methods are contemplated. Intravenous injection to the portal veinis a preferred method of administration. Other in vivo administrationmodes include, for example, direct injection into the lobes of the liveror the biliary duct and intravenous injection distal to the liver,including through the hepatic artery, direct injection in to the liverparenchyma, injection via the hepatic artery, and/or retrogradeinjection through the biliary tree. Ex vivo modes of administrationinclude transduction in vitro of resected hepatocytes or other cells ofthe liver, followed by infusion of the transduced, resected hepatocytesback into the portal vasculature, liver parenchyma or biliary tree ofthe human patient, see e.g., Grossman et al. (1994) Nature Genetics6:335-341. Other modes of administration include the ex vivonuclease-mediated insertion of a Factor VII, Factor VIII, Factor IX,Factor X and/or Factor XI encoding transgene into a safe harbor locationinto patient or allogenic stem cells. Following modification, thetreated cells are then re-infused into the patient for treatment of ahemophilia.

Treatment of hemophilias is discussed herein by way of example only, andit should be understood that other conditions may be treating using themethods and compositions disclosed herein. In some cases, the methodsand composition may be useful in the treatment of thrombotic disorders.The effective amount of nuclease(s) and Factor VII, Factor VIII, FactorIX, Factor X, or Factor XI donor to be administered will vary frompatient to patient and according to the therapeutic polypeptide ofinterest. Accordingly, effective amounts are best determined by thephysician administering the compositions and appropriate dosages can bedetermined readily by one of ordinary skill in the art. After allowingsufficient time for integration and expression (typically 4-15 days, forexample), analysis of the serum or other tissue levels of thetherapeutic polypeptide and comparison to the initial level prior toadministration will determine whether the amount being administered istoo low, within the right range or too high. In some embodiments, asingle dose may be administered to a patient. In some embodiments,multiple doses may be administered to a patient. In some embodiments,co-administration of the compositions disclosed herein with othertherapeutic agents may be performed.

Suitable regimes for initial and subsequent administrations are alsovariable, but are typified by an initial administration followed bysubsequent administrations if necessary. Subsequent administrations maybe administered at variable intervals, ranging from daily to annually toevery several years. One of skill in the art will appreciate thatappropriate immunosuppressive techniques may be recommended to avoidinhibition or blockage of transduction by immunosuppression of thedelivery vectors, see e.g., Vilquin et al. (1995) Human Gene Ther.6:1391-1401.

Formulations for both ex vivo and in vivo administrations includesuspensions in liquid or emulsified liquids. The active ingredientsoften are mixed with excipients which are pharmaceutically acceptableand compatible with the active ingredient. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like, andcombinations thereof. In addition, the composition may contain minoramounts of auxiliary substances, such as, wetting or emulsifying agents,pH buffering agents, stabilizing agents or other reagents that enhancethe effectiveness of the pharmaceutical composition.

The following Examples relate to exemplary embodiments of the presentdisclosure in which the nuclease comprises a zinc finger nuclease (ZFN).It will be appreciated that this is for purposes of exemplification onlyand that other nucleases can be used, for instance homing endonucleases(meganucleases) with engineered DNA-binding domains and/or fusions ofnaturally occurring of engineered homing endonucleases (meganucleases)DNA-binding domains and heterologous cleavage domains, TALENs and/or aCRISPR/Cas system comprising an engineered single guide RNA.

EXAMPLES Example 1: Nuclease and Donor Construction

To target the human albumin locus two individual ZFNs, SBS42906(5-finger protein) and SBS43043 (6-finger protein) were designed to bindadjacent 15 base pair and 18 base pair target sites, respectively, withhigh affinity and specificity. The ZFNs are shown below in Table 1.

Uppercase in the target sequence denotes bound nucleotides and lowercasedenotes unbound nucleotides.

TABLE 1 Albumin specific ZFNs Design Albumin specific ZFNs SBS #, TargetF1 F2 F3 F4 F5 F6 SBS# 42906 QSGNLAR LKQNLCM WADNLQN TSGNLTR RQSHLCL NAttTGGGATAGTTA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID TGAAttcaatcttNO: 4) NO: 5) NO: 6) NO: 7) NO: 8) ca (SEQ ID NO: 2) SBS#43043 TPQLLDRLKWNLRT DQSNLRA RNFSLTM LRHDLDR HRSNLNK ccTATCCATTGCA (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID CTATGCTttattt NO: 9) NO: 10) NO: 11)NO: 12) NO: 13) NO: 14) aa (SEQ ID NO: 3)

The strict requirement of both ZFNs being bound to a combined 33 basepair recognition sequence in a specific spatial orientation on the DNAprovides the functional specificity necessary to catalyze the formationof a double-strand break (DSB) at a single pre-determined site in thealbumin locus. A schematic representation of this architecture is shownin FIG. 2 . The two ZFNs are each targeted to a specific sequence in anintronic region of the albumin gene (nucleotides 447-485 relative to thetranscription start site) located on opposite strands of DNA.

A FokI nuclease cleavage domain is attached to the carboxy-terminal endof each ZFP. See, e.g., U.S. Pat. Nos. 7,888,121 and 8,623,618. Anadditional feature of the FokI domain that enforces highly specificfunction is the restriction of cleavage only to heterodimer bindingevents. This is achieved through the use of variant FokI domains (e.g.,“ELD/KKR”) that have been engineered to function only as heterodimers.See, e.g., U.S. Pat. No. 8,623,618. These obligate heterodimer Folddomains prevent off-target cleavage at potential homodimer sites, thusfurther enhancing ZFN specificity.

Donor vectors were constructed as follows. The rAAV2/6 vectors encodingthe ZFNs targeting the albumin intron 1 locus and hF9 transgene areshown in FIG. 1 . For the two ZFN encoding AAV2/6 vectors, SB-42906 andSB-43043 (expressing the left and right ZFN respectively), ZFNexpression is under control of a liver-specific enhancer and promoter,comprised of the human ApoE enhancer and human α1-anti-trypsin (hAAT)promoter (Miao et al. (2000) Mol. Ther. 1(6):522-532). The ApoE/hAATpromoter is highly active in hepatocytes, the intended target tissue,but is inactive in other cell and tissue types to prevent ZFN expressionand activity in non-target tissues.

The rAAV2/6 donor vector containing the hF9 transgene (SB-F9 donor) is apromoterless construct that encodes a partial hF9 cDNA comprising exons2-8. The F9 exon 2 splice acceptor site (SA) is present to allowefficient splicing of hF9 transgene (exons 2-8) into the mature mRNAfrom the albumin locus, regardless of the mechanism of integration (NHEJor HDR; see, e.g., FIGS. 3 and 4 ). Optionally flanking the hF9transgene are sequences homologous to the cleavage site at the albuminintron 1 locus. The left arm of homology (LA) contains 280 nucleotidesof identical sequence upstream of the albumin intron 1 cleavage site,and the right arm of homology (RA) contains 100 nucleotides of identicalsequence downstream of the cleavage site. The arms of homology are usedto help facilitate targeted integration of the hF9 transgene at thealbumin intron 1 locus via homology-directed repair. The sizes of thehomology arms were chosen to avoid repetitive sequences and splicingelements in the albumin locus that can inhibit targeted integration ortransgene expression. The PolyA sequences are derived from the BovineGrowth Hormone gene. The rAAV vectors are packaged with capsid serotypeAAV2/6 using a Baculovirus (Sf9) expression system.

Example 2: Nuclease Modification in Human Primary Hepatocytes

One day after plating, human primary hepatocytes were transduced witheither one or two ZFN expressing rAAV2/6 virus(es) at an MOI of0.3E5-9.0E5 vector genomes (vg) per cell. Cells were harvested at Day 7and genomic DNA and protein were prepared for both percent indels(insertions and/or deletions) analysis by miSEQ and ZFN expressionanalysis by Western blot using an antibody against the FokI domain.

The ZFNs described herein (SBS42906 (5-finger; left ZFN) and SBS43043(6-finger; right ZFN)) exhibited high levels of modification (e.g.,between 10-30% indels).

In addition, a human hepatoma cell line (HepG2) was used to evaluatespecific nuclease-mediated integration at the albumin locus and mode ofintegration (NHEJ or HDR). HepG2 cells transduced with human albuminZFNs and FIX donor showed strong hFIX secretion (˜75 ng/ml) abovebackground, and analysis of albumin-specific gene modification by miSEQshowed ˜74% indels. Subclones were used for genotyping to determine thespecific integration at the albumin locus and the mode of integrationeither by NHEJ or HDR (FIG. 3 ). Subclones also showed strong hFIXsecretion (up to 350 ng/ml). Overall, there was no difference betweensubclones with monoallelic donor integration by either HDR or NHEJ.

These results showed that hFIX can be stably expressed from the albuminlocus independent of the mode of FIX donor integration.

After stable integration of the FIX donor at the albumin locus, thehALB-hFIX fusion protein mRNA is transcribed from the albumin promoter.While expression of the unaltered wild type Albumin gene leads to a 2.3kb transcript, the hALB-hFIX fusion mRNA is shorter with 1.7 kb (FIG.5A) independently of the integration mode by either NHEJ or HDR.

We demonstrated this by creating stable subclones of the Hepatoma cellline HepG2 with stably integrated FIX donors either by NHEJ or HDR.After transduction of HepG2 cells with SB-42906, SB-43043 and FIX donorwe identified by integration-specific genotyping several subclones,which exhibited FIX donor integration by either NHEJ or HDR and whichshowed high (150-350 ng/ml) secreted levels of hFIX proteins detectableby hFIX ELISA (FIG. 5B). After total mRNA isolation and reversetranscription to cDNA we analyzed the transcripts of these subclones byPCR and sequencing. In unmodified control cells we could only detect theexpected wild type Albumin transcript (2.3 kb) with primers flanking theexon 1-8, but no transcript with primers binding to the FIX donor.

In three HepG2 subclones that have the FIX donor integrated into thealbumin locus and secrete FIX, we detected both the wild type humanalbumin transcript and with primers binding to the Albumin 5′UTR and theFIX donor 3′UTR the expected hALB-hFIX fusion transcript (1.7 kb).Subcloning and sequencing of these RT-PCR products confirmed that it isthe expected hAlb-hFIX fusion mRNA. This suggests that in all threesubclones the FIX donor integrated either by NHEJ or HDR in one Albuminallele which led to the expression of a functional hALB-hFIX transcriptand further secreted hFIX protein. MiSeq analysis of the other alleleshowed that these subclones carried a 10 or 40 nucleotide (nt) deletionor a 1 nt insertion in albumin intron1. Despite these indels, the cellsas expected produce about half the amount of wild-type albumintranscript and protein as the unmodified control cells, demonstratingthat small indels in albumin exon1 have no major impact on albuminexpression. Since in these subclones there were no other transcriptsdetectable with primers binding to exon8 of the albumin mRNA, weconcluded that after hFIX donor integration mRNA splicing occurs onlybetween the splice donor site of the Albumin exon1 and the spliceacceptor site of the hF9 donor.

In summary, our detailed analysis of the transcription profile at thenuclease-modified albumin locus in HepG2 subclones showed that only thedesired hALB-hFIX fusion protein mRNA is expressed.

Successful targeted integration requires all three AAV vectors totransduce the same hepatocyte (see for example, U.S. Provisional PatentApplication No. 61/943,865). We have found that the efficiency oftargeted integration is dependent on the ratio of ZFN:Donor. Thus, therAAV-encoded ZFN vectors are formulated at a 1:1 ratio, and therAAV-encoded ZFN:ZFN:hF9 donor DNA ratio is 1:1:8.

Example 3: In Vitro Off-Target SELEX-Guided Toxicity Evaluation inHumans

A bioinformatics approach was used to identify potential off-targeteffects of ZFN action by searching the genome for best-fit matches tothe intended DNA binding sites for the ZFNs as described herein. Toobtain a consensus preference binding site for each ZFN, anaffinity-based target site selection procedure known as SELEX (systemicevolution of ligands by experimental enrichment) was employed.

Briefly, the SELEX experiment was performed by incubating the ZFPportion of the ZFN with a pool of random DNA sequences, capturing theZFP protein and any bound DNA sequences via affinity chromatography, andthen PCR amplifying the bound DNA fragments. These DNA fragments werecloned and sequenced and aligned to determine the consensus bindingpreference.

These experimentally derived preferences for every position in the 15base pair and 18 base pair binding sites for the two ZFNs of Table 1were then used to guide a genome-wide bioinformatics search for the mostsimilar sites in the human genome. The resulting list of potentialcleavage sites was then ranked to give priority to those sites with thehighest similarity to the SELEX-derived consensus sequences. The top 40sites in the human genome were identified. Of these 40 sites, 18 fellwithin annotated genes, but only 1 of these occurred within exonic(protein coding) sequences. Low levels of modification at an intronicsequence would not be expected to impact gene expression or function.

Next, to determine if these sites are cleaved by the nucleases describedherein, human primary hepatocytes and the human hepatoma cell line HepG2were transduced with high doses (3e5 vg/cell) of SB-42906 and SB-43043(both packaged as AAV2/6) or an AAV2/6 vector expressing GFP as control.Genomic DNA was isolated 7-9 days post-transfection, and the on-target(hALB intron 1 locus) and top 40 predicted off-target site loci were PCRamplified from 100 ng of genomic DNA (˜15,000 diploid genomes). Thelevel of modification at each locus was then determined by paired-enddeep sequencing on an Illumina miSEQ. Paired sequences were merged viathe open source software package SeqPrep (developed by Dr. John St.John, for example see Klevegring et al. (2014) PLOS One doi: e104417doi.10,1371journal.pone 0104417).

Each sequence was filtered for a quality score of ≥15 across all bases,then mapped to the human genome (hg19 assembly). Sequences which mappedto an incorrect locus were removed from further analysis. ANeedleman-Wunsch (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol Biol48(3):443-53) alignment was performed between the target amplicon genomeregion and the obtained Illumina Miseq read to map insertions anddeletions. Indel events in aligned sequences were defined as describedin Gabriel et al. (2011) Nature Biotechnology. 29(9):816-23 except thatindels 1 base pair in length were also considered true indels to avoidundercounting real events.

After application of the described analysis the on-target modificationwas measured at 16.2% indels in human primary hepatocytes and 30.0%indels in HepG2 cells. No significant indels could be detected at any ofthe potential top 40 off-target sites in either cell type.

These results highlight the exquisite specificity of the ZFN reagentsSBS42906 and SBS43043 in both human primary hepatocyte and hepatomacells at on-target levels (16 and 30%, respectively).

Example 4: In Vivo Targeted Integration

The nucleases (and/or polynucleotides encoding the nucleases) and donorsand/or pharmaceutical compositions comprising the nucleases and/ordonors as described herein are administered to a human subject such thata corrective FIX transgene is integrated into the subject's genome(e.g., under the control of, the subject's own endogenous albuminlocus), thus resulting in liver-specific synthesis of Factor IX. Inparticular, male subjects, at least 18 years of age, with severeHemophilia B (>6 hemorrhages/year when untreated) who are receivingprophylactic FIX replacement therapy (>100 doses) as per currenttreatment guideline without inhibitors to FIX and have nohypersensitivity to recombinant FIX are administered nucleases and FIXdonors as described herein according to one of the following schedulesillustrated in Table 2:

TABLE 2 ZFN 1 ZFN 2 cDNA Donor Total rAAV Dose (vg/kg) (vg/kg) (vg/kg)(vg/kg) 5.00E+11 5.00E+11 4.00E+12 5.00E+12 1.00E+12 1.00E+12 8.00E+121.00E+13 5.00E+12 5.00E+12 4.00E+13 5.00E+13 1.00E+13 1.00E+13 8.00E+131.00E+14 * ZFN1:ZFN2:cDNA Donor Ratio used: 1:1:8 ZFN1:ZFN2:cDNA Donorratio

“SB-FIX” contains three components, namely three individual recombinantadeno-associated virus (rAAV) serotype 2/6 (rAAV2/6) vectors,administered as an intravenous dose: a first vector (SB-42906) encodingSBS42906 (designated the left ZFN and herein referred to as ZFN1), asecond vector (SB-43043) encoding SBS43043 (designated the right ZFN andherein referred to as ZFN2), and a third vector (hF9 gene donor)encoding a DNA repair template encoding a promoterless hF9 transgene.

The 3 components of SB-FIX (2 nucleases and donor) are administeredsequentially and/or concurrently, for example in one or morepharmaceutical compositions, via intravenous infusion (e.g., portalvein).

Optionally subjects are treated (before, during and/or after SB-FIXadministration) with immunosuppressive agents such as corticosteriods,for example to reduce or eliminate immune responses (e.g., neutralizingantibody (NAB) responses or immune responses to AAV). In particular,subjects who develop increased liver aminotransferases will beadministered a brief immunosuppressive regimen with 60 mg ofprednisolone followed by a taper over 4-6 weeks.

Subjects are evaluated for an immune response to AAV2/6; an immuneresponse to FIX; the presence of AAV2/6 vector DNA; the presence ofalbumin locus gene modifications (modification site of SB-FIX) by PCR inblood, saliva, urine, stool and semen; change from baseline in FIXlevels; change from baseline in aPTT; any change from baseline in use ofFactor IX replacement therapy; any change from baseline in frequencyand/or severity of bleeding episodes; and liver function (including, forexample, AST, ALT, bilirubin, alkaline phosphatase, and albumin levels).

SB-FIX administration restores hepatic production of functional FIX.At >1% of normal levels, the need for prophylactic treatment with FIXconcentrate is reduced or eliminated and at >5% of normal, hemorrhagefollowing all but the most severe trauma is substantially reduced.AAV-mediated gene transfer of the FIX cDNA into liver cells thusprovides long term production of FIX in Hemophilia B subjects.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A cell comprising a sequence encoding a Factor IX(F.IX) protein integrated into intron 1 of an endogenous albumin genesuch that the F.IX protein is expressed from the albumin gene.
 2. Thecell of claim 1, wherein the cell is a liver cell.
 3. A method oftreating a patient with hemophilia B, the method comprising generating acell according to claim 2 in the patient, thereby treating the patient.4. The method of claim 3, wherein the cell is generated in the patientby administering at least one polynucleotide comprising sequencesencoding a nuclease and the F.IX protein.
 5. The method of claim 4,wherein the nuclease comprises a zinc finger nuclease (ZFN), aTAL-effector domain nuclease (TALEN) or a CRISPR/Cas nuclease system. 6.The method of claim 5, wherein the nuclease comprises a ZFN comprisingfirst and second zinc finger nucleases, the first zinc finger nucleasecomprising a FokI cleavage domain and a zinc finger protein comprising 5zinc finger domains ordered F1 to F5, wherein each zinc finger domaincomprises a recognition helix region and wherein the recognition helixregions of the zinc finger protein are SEQ ID NOs: 4, 5, 6, 7, and 8,and the second zinc finger nuclease comprising a FokI cleavage domainand a zinc finger protein comprising 6 zinc finger domains ordered F1 toF6, wherein each zinc finger domain comprises a recognition helix regionand wherein the recognition helix regions of the zinc finger protein areSEQ ID NOs: 9, 10, 11, 12, 13, and
 14. 7. The method of claim 4, whereinthe at least one polynucleotide encoding the nuclease and the F.IXprotein is administered intravenously to the patient.
 8. The method ofclaim 4, wherein first, second and third polynucleotides areadministered to the patient, the first polynucleotide encoding a firstzinc finger nuclease, the second polynucleotide encoding a second zincfinger protein and the third polynucleotide comprising the sequenceencoding the F.IX protein and further wherein the first and second zincfinger nuclease dimerize to form a zinc finger nuclease.
 9. The methodof claim 8, wherein the first, second and third polynucleotides arecarried on AAV vectors.
 10. The method of claim 8, wherein the first,second and third polynucleotides are administered in a ratio of about1:1:1, about 1:1:2, about 1:1:3, about 1:1:4, about 1:1:5, about 1:1:6,about 1:1:7, about 1:1:8, about 1:1:9, about 1:1:10, about 1:1:11, about1:1:12, about 1:1:13, about 1:1:14, about 1:1:15, about 1:1:16, about1:1:17, about 1:1:18, about 1:1:19, or about 1:1:20.
 11. The method ofclaim 9, wherein the AAV vectors are administered in a ratio about1:1:1, about 1:1:2, about 1:1:3, about 1:1:4, about 1:1:5, about 1:1:6,about 1:1:7, about 1:1:8, about 1:1:9, about 1:1:10, about 1:1:11, about1:1:12, about 1:1:13, about 1:1:14, about 1:1:15, about 1:1:16, about1:1:17, about 1:1:18, about 1:1:19, or about 1:1:20.